Recombinant 47 and 31KD cocoa proteins and precursor

ABSTRACT

PCT No. PCT/GB91/00914 Sec. 371 Date Jan. 21, 1993 Sec. 102(e) Date Jan. 21, 1993 PCT Filed Jun. 7, 1991 PCT Pub. No. WO91/19801 PCT Pub. Date Dec. 26, 199147 kD and 31 kD proteins, and their 67 kD expression precursor, believed to be the source of peptide flavour precursors in cocoa (Theobroma cacao) have been identified. Genes coding for them have been probed, identified and sequenced, and recombinant proteins have been synthesised.

This application is filed under 35 USC §371 as the national stage of PCT/G891/00914 filed Jun. 7, 1991.

This invention relates to proteins and nucleic acids derived from or otherwise related to cocoa.

The beans of the cocoa plant (Theobroma cacao) are the raw material for cocoa, chocolate and natural cocoa and chocolate flavouring. As described by Rohan ("Processing of Raw Cocoa for the Market", FAO/UN (1963)), raw cocoa beans are extracted from the harvested cocoa pod, from which the placenta is normally removed, the beans are then "fermented" for a period of days, during which the beans are killed and a purple pigment is released from the cotyledons. During fermentation "unknown" compounds are formed which on roasting give rise to characteristic cocoa flavour. Rohan suggests that polyphenols and theobromine are implicated in the flavour precursor formation. After fermentation, the beans are dried, during which time the characteristic brown pigment forms, and they are then stored and shipped.

Biehl et al. 1982 investigated proteolysis during anaerobic cocoa seed incubation and identified 26 kD and 44 kD proteins which accumulated during seed ripening and degraded during Germination. Biehl asserted that there were storage proteins and suggested that they may give rise to flavour-specific peptides.

Fritz et al, 1985 identified polypeptides of 20 kD and 28 kD appearing in the cytoplasmic fraction of cocoa seed extracts at about 100 days after pollination. It appears that the 20 kD protein is thought to have glyceryl acyltransferase activity.

In spite of the uncertainties in the art, as summarised above, proteins apparently responsible for flavour production in cocoa beans have now been identified. Further, it has been discovered that, in spite of Fritz's caution that "cocoa seed mRNA levels are notably low compared to other plants" (loc. cit.), it is possible to apply the techniques of recombinant DNA techniques to the production of such proteins.

According to a first aspect of the invention, there is provided a 67 kD protein of Theobroma cacao, or a fragment thereof.

The 67 kD protein appears to be a primary translation product of interest in proteins involved in flavour production in cocoa. The 67 kD protein may be processed in vivo to form 47 kD and 31 kD polypeptides.

According to a second aspect of the invention, there is provided a 47 kD protein of Th. cacao, or a fragment thereof.

According to a third aspect of the invention, there is provided a 31 kD protein of Th. cacao or a fragment thereof.

The term "fragment" as used herein and as applied to proteins or peptides indicates a sufficient number of amino acid residues are present for the fragment to be useful. Typically, at least four, five, six or even at least 10 or 20 amino acids may be present in a fragment. Useful fragments include those which are the same as or similar or equivalent to those naturally produced during the fermentation phase of cocoa bean processing. It is believed that such fragments take part in Maillard reactions during roasting, to form at least some of the essential flavour components of cocoa.

Proteins in accordance with the invention may be synthetic; they may be chemically synthesised or, preferably, produced by recombinant DNA techniques. Proteins produced by such techniques can therefore be termed "recombinant proteins". Recombinant proteins may be glycosylated or non-glycosylated: non-glycosylated proteins will result from prokaryotic expression systems. Theobroma cacao has two primary subspecies, Th. cacao cacao and Th. cacao sphaerocarpum. While proteins in accordance with the invention may be derived from these subspecies, the invention is not limited solely to these subspecies. For example, many cocoa varieties are hybrids between different species; an example of such a hybrid is the trinitario variety.

The invention also relates to nucleic acid, particularly DNA, coding for the proteins referred to above (whether the primary translation products, the processed proteins or fragments). The invention therefore also provides, in further aspects:

nucleic acid coding for a 67 kD protein of Th. cacao, or for a fragment thereof:

nucleic acid coding for a 47 kD protein of Th. cacao, or for a fragment thereof;

nucleic acid coding for a 31 kD protein of Th. cacao, or for a fragment thereof:

Included in the invention is nucleic acid which is degenerate for the wild type protein and which codes for conservative or other non-deleterious mutants. Nucleic acid which hybridises to the wild type material is also included.

Nucleic acid within the scope of the invention will generally be recombinant nucleic acid and may be in isolated form. Frequently, nucleic acid in accordance with the invention will be incorporated into a vector (whether an expression vector or otherwise) such as a plasmid. Suitable expression vectors will contain an appropriate promoter, depending on the intended expression host. For yeast, an appropriate promoter is the yeast pyruvate kinase (PK) promoter; for bacteria an appropriate promoter is a strong lambda promoter.

Expression may be secreted or non-secreted. Secreted expression is preferred, particularly in eukaryotic expression systems; an appropriate signal sequence may be present for this purpose. Signal sequences derived from the expression host (such as that from the yeast alpha-factor in the case of yeast) may be more appropriate than native cocoa signal sequences.

The invention further relates to host cells comprising nucleic acid as described above. Genetic manipulation may for preference take place in prokaryotes. Expression will for preference take place in a food-approved host. The yeast Saccharomyces cerevisiae is particularly preferred.

The invention also relates to processes for preparing nucleic acid and protein as described above by nucleic acid replication and expression, respectively.

cDNA in accordance with the invention may be useful not only for obtaining protein expression but also for Restriction Fragment Length Polymorphism (RFLP) studies. In such studies, detectably labelled cDNA (eg radiolabelled) is prepared. DNA of a cultivar under analysis is then prepared and digested with restriction enzymes. Southern blotting with the labelled cDNA may then enable genetic correlations to be made between cultivars. Phenotypic correlations may then be deduced.

The invention will now be illustrated by the following non-limiting examples. The examples refer to the accompanying drawings, in which: FIG. 1 shows a map of the coding region of the 67 kD protein, together with the inter-relationship of plasmids pMS600, pMS700 and pMS800, from which sequence data were obtained;

FIGS. 2A-2D (SEQ ID NOS: 1 and 2) shows the complete nucleotide sequence of cDNA coding for the 67 kD protein and the deduced amino acid sequence;

FIG. 3 (SEQ ID NO:22) shows the amino acid sequence referred to in FIG. 2;

FIGS. 4A-4D (SEQ ID NOS: 22-27) shows the relationship between the 67 kD protein and seed storage proteins from other plants;

FIG. 5 (SEQ ID NO:28) shows a map of plasmid pJLA502;

FIG. 6 shows schematically the formation of plasmid pMS900;

FIG. 7 shows two yeast expression vectors useful in the present invention; vector A is designed for internal expression and vector B is designed for secreted expression;

FIG. 8a (SEQ ID NOS:4-11) shows, in relation to vector A, part of the yeast pyruvate kinase gene showing the vector A cloning site, and the use of Hin-Nco linkers to splice in the heterologous gene;

FIG. 8b (SEQ ID NOS:12-17) shows, in relation to vector B, part of the yeast alpha-factor signal sequence showing the vector B cloning site, and the use of Hin-Nco linkers to create an in-phase fusion;

FIG. 9a shows how plasmid pMS900 can be manipulated to produce plasmids pMS901, pMS903, pMS907, pMS908, pMS911, pMS912 and pMS914;

FIG. 9b shows how plasmid pMS903 can be manipulated to produce plasmids pMS904, pMS905, pMS906, pMS909 and pMS916;

FIG. 10 shows maps of plasmids pMS908, pMS914, pMS912, pMS906, pMS916 and pMS910;

FIG. 11 shows the construction of a plasmid to express the 67 kD protein by means of the AOX promoter on an integrated vector in Hansenula polymorpha; and

FIG. 12 shows the construction of a plasmid to express the 67 kD protein by means of the AOX promoter in conjunction with the yeast α-factor secretory signal on an integrated vector in Hansenula polymorpha.

EXAMPLES Example 1

Identification of the Major Seed Proteins

It is not practicable to extract proteins directly from cocoa beans due to the high fat and polyphenol contents, and proteins were, therefore, extracted from acetone powders made as follows. Mature beans from cocoa of West African origin (Theobroma cacao amelonada) were lyophilised and ground roughly in a pestle and mortar. Lipids were extracted by Soxhlet extraction with diethyl ether for two periods of four hours, the beans being dried and further ground between extractions. Polyphenols and pigments were then removed by several extractions with 80% acetone. 0.1% thioglycollic acid. After extraction the resulting paste was dried under vacuum and ground to a fine powder.

Total proteins were solubilised by grinding the powder with extraction buffer (0.05M sodium phosphate. pH 7.2; 0.01M 2-mercaptoethanol; 1% SDS) in a hand-held homogeniser, at 5 mg/ml The suspension was heated at 95° C. for 5 minutes, and centrifuged at 18K for 20 minutes to remove insoluble material. The resulting clear supernatant contained about 1 mg/ml total protein. Electrophoresis of 25 μl on an SDS-PAGE gel (Laemmli, 1970) gave three major bands, two of which were at 47 kD and 31 kD, comprising over 60% of the total proteins. The 47 kD and 31 kD proteins are presumed to be the polypeptide subunits of major storage proteins.

Characteristics of the Storage Polypeptides

The solubility characteristics of the 47 kD and 31 kD polypeptides were roughly defined by one or two quick experiments. Dialysis of the polypeptide solution against SDS-free extraction buffer rendered the 47 kD and 31 kD polypeptides insoluble, as judged by their ability to pass through a 0.22 micron membrane. Fast Protein Liquid Chromatography (FPLC) analysis also showed that the 47 kD and 31 kD polypeptides were highly associated after extraction with Mcllvaines buffer pH 6.8 (0.2M disodium hydrogen phosphate titrated with 0.1M citric acid). The 47 kD and 31 kD polypeptides are globulins on the basis on their solubility.

Purification of the 47 kD and 31 kD polypeptides

The 47 kD and 31 kD polypeptides were purified by two rounds of gel filtration on a SUPEROSE-12 column of the PHARMACIA Fast Protein Liquid Chromatography system (FPLC), or by electroelution of bands after preparative electrophoresis. (The words SUPEROSE and PHARMACIA are trade marks.) Concentrated protein extracts were made from 50 mg acetone powder per ml of extraction buffer, and 1-2 ml loaded onto 2 mm thick SDS-PAGE gels poured without a comb. After electrophoresis the gel was surface stained in aqueous Coomassie Blue, and the 47 kD and 31 kD bands cut out with a scalpel. Gel slices were electroeluted into dialysis bags in electrophoresis running buffer at 15 V for 24 hours, and the dialysate dialysed further against 0.1 % SDS. Samples could be concentrated by lyophilisation.

Example 2

Amino-acid Sequence Data from Proteins

Protein samples (about 10 μg) were subjected to conventional N-terminal amino-acid sequencing. The 47 kD and 31 kD polypeptides were N-terminally blocked, so cyanogen bromide peptides of the 47 kD and 31 kD peptides were prepared, and some amino-acid sequence was derived from these. Cyanogen bromide cleaves polypeptide chains at methionine residues, and thus cleaved the 47 kD and 31 kD polypeptides gave rise to 24 kD and 17 kD peptides. In addition the 47 kD polypeptide gave a 20 kD peptide. The 24 kD and 17 kD peptides had the same 9 N-terminal amino-acid residues. This fact, combined with the obvious one that the 31 kD could not contain both peptides consecutively, suggested that the 24 kD peptide arose for a partial digest, where full digestion would yield the 17 kD peptide. The other striking conclusion is that the 47 kD and 31 kD proteins are related, and the 31 kD could be a further processed form of the 47 kD. The 9 amino-acid sequence was used to construct an oligonucleotide probe for the 47 kD/3 1 kD gene(s).

Example 3

Raising Antibodies to the 47 kD and 31 kD Polypeptides

Polyclonal antibodies were prepared using the methodology of Catty and Raykundalia (1988). The serum was aliquoted into 1 ml fractions and stored at -20° C.

Characterising Antibodies to the 47 kD and 31 kD Polypeptides

Serum was immediately characterised using the Ochterloney double-diffusion technique, whereby antigen and antibody are allowed to diffuse towards one another from wells cut in agarose in borate-saline buffer. Precipitin lines are formed where the two interact if the antibody `recognises` the antigen. This test showed that antibodies to both antigens had been formed, and furthermore that extensive cross-reaction took place between the 47 kD and 31 kD polypeptides and their respective antibodies. This is further indication that the 47 kD and 31 kD polypeptides are closely related, as suggested by their cyanogen bromide cleavage patterns.

The gamma-globulin fraction of the serum was partially purified by precipitation with 50% ammonium sulphate, solubilisation in phosphate-buffered saline (PBS) and chromatography on a DE 52 cellulose ion-exchange column as described by Hill, 1984. Fractions containing gamma-globulin were monitored at 280 nm (OD₂₈₀ of 1.4 is equivalent to 1 mg/ml gamma-globulin) and stored at -20° C. The effective titre of the antibodies was measured using an enzyme-linked immunosorbant assay (ELISA). The wells of a polystyrene microtitre plate were coated with antigen (10-1000 ng) overnight at 4° C. in carbonate coating buffer. Wells were washed in PBS-Tween and the test gamma globulin added at concentrations of 10, 1 and 0.1 μg/ml (approximately 1:100, 1:1000 and 1:10,000 dilutions). The diluent was PBS-Tween containing 2% polyvinyl pyrrolidone (PVP) and 0.2% BSA. Controls were preimmune serum from the same animal. Binding took place at 37° C. for 3-4 hours. The wells were washed as above and secondary antibody (goat anti-rabbit IgG conjugated to alkaline phosphatase) added at a concentration of 1 μg/ml, using the same conditions as the primary antibody. The wells are again washed, and alkaline phosphatase substrate (p-nitrophenyl phosphate pH 0.6 mg/ml in diethanol-amine buffer pH 9.8) added. The yellow colour, indicating a positive reaction, was allowed to develop for 30 minutes and the reaction stopped with 3M NaOH. The colour is quantified at 405 nm. More detail of this method is given in Hill, 1984. The method confirmed that the antibodies all had a high titre and could be used at 1 μg/ml concentration.

Example 4

Isolation of Total RNA from Immature Cocoa Beans

The starting material for RNA which should contain a high proportion of mRNA specific for the storage proteins was immature cocoa beans, at about 130 days after pollination. Previous work had suggested that synthesis of storage proteins was approaching its height by this date (Biehl et al, 1982). The beans are roughly corrugated and pale pinkish-purple at this age.

The initial requirement of the total RNA preparation from cocoa beans was that it should be free from contaminants, as judged by the UV spectrum, particularly in the far UV, where a deep trough at 230 nm (260 nm:230 nm ratio is approximately 2.0) is highly diagnostic of clean RNA, and is intact, as judged by agarose gel electrophoresis of heat-denatured samples, which should show clear rRNA bands. A prerequisite for obtaining intact RNA is scrupulous cleanliness and rigorous precautions against RNases, which are ubiquitous and extremely stable enzymes. Glassware is customarily baked at high temperatures, and solutions and apparatus treated with the RNase inhibitor diethyl pyrocarbonate (DEPC, 0.1%) before autoclaving.

The most routine method for extraction of plant (and animal) RNA is extraction of the proteins with phenol/chloroform in the presence of SDS to disrupt protein-nucleic acid complexes, and inhibit the RNases which are abundant in plant material. Following phenol extraction the RNA is pelletted on a caesium chloride gradient before or after ethanol precipitation. This method produced more or less intact RNA, but it was heavily contaminated with dark brown pigment, probably oxidised polyphenols and tannins, which always co-purified with the RNA. High levels of polyphenols are a major problem in Theobroma tissues.

A method was therefore adopted which avoided the use of phenol, and instead used the method of Hall et al. (1978) which involves breaking the tissue in hot SDS-borate buffer, digesting the proteins with proteinase K, and specifically precipitating the RNA with LiCl. This method gave high yields of reasonably clean, intact RNA. Contaminants continued to be a problem and the method was modified by introducing repeated LiCl precipitation steps, the precipitate being dissolved in water and clarified by microcentrifugation after each step. This resulted in RNA preparations with ideal spectra, which performed well in subsequent functional tests such as in vitro translation.

Preparation of mRNA From Total RNA

The mRNA fraction was separated from total RNA by affinity chromatography on a small (1 ml) oligo-dT column, the mRNA binding to the column by its poly A tail. The RNA (1-2 mg) was denatured by heating at 65° C. and applied to the column in a high salt buffer. Poly A+was eluted with low salt buffer, and collected by ethanol precipitation. The method is essentially that of Aviv and Leder (1972), modified by Maniatis et al (1982). From 1 mg of total RNA, approximately 10-20 g polyA+RNA was obtained (1-2%).

In vitro Translation of mRNA

The ability of mRNA to support in vitro translation is a good indication of its cleanliness and intactness. Only mRNAs with an intact polyA tail (3' end) will be selected by the oligo-dT column, and only mRNAs which also have an intact 5' end (translational start) will translate efficiently. In vitro translation was carried out using RNA-depleted wheat-germ lysate (Amersham International), the de novo protein synthesis being monitored by the incorporation of ³⁵ S!-methionine (Roberts and Paterson, 1973). Initially the rate of de novo synthesis was measured by the incorporation of ³⁵ S!-methionine into TCA-precipitable material trapped on glass fibre filters (GFC, Whatman). The actual products of translation were investigated by running on SDS-PAGE, soaking the gel in fluor, drying the gel and autoradiography. The mRNA preparations translated efficiently and the products covered a wide range of molecular weights, showing that intact mRNAs for even the largest proteins had been obtained. None of the major translation products corresponded in size to the 47 kD or 3 kD storage polypeptides identified in mature beans, and it was apparent that considerable processing of the nascent polypeptides must occur to give the mature forms.

Example 5

Identification of Precursor to the 47 kD and 31 kD Polypeptides by Immunoprecipitation

Because the 47 kD and 31 kD storage polypeptides were not apparent amongst the translation products of mRNA from developing cocoa beans, the technique of immunoprecipitation, with specific antibodies raised to the storage polypeptides, was used to identify the precursors from the translation mixture. This was done for two reasons: first to confirm that the appropriate mRNA was present before cloning, and second to gain information on the expected size of the encoding genes.

Immunoprecipitation was by the method of Cuming et al, 1986. ³⁵ S!-labelled in vitro translation products were dissociated in SDS, and allowed to bind with specific antibody in PBS plus 1% BSA. The antibody-antigen mixture was then mixed with protein A-SEPHAROSE and incubated on ice to allow the IgG to bind to protein A. The slurry was poured into a disposable 1 ml syringe, and unbound proteins removed by washing with PBS+1% NONIDET P-40. The bound antibody was eluted with 1M acetic acid and the proteins precipitated with TCA. The antibody-antigen complex was dissociated in SDS, and subject to SDS-PAGE and fluorography, which reveals which labelled antigens have bound to the specific antibodies.

The results showed that the anti-47 kD and anti-31 kD antibodies both precipitated a 67 kD precursor. The precursor size corresponded to a major band on the in vitro translation products. The results with the 47 kD and 31 kD antibodies confirmed that the polypeptides are derived from a single precursor, or at least precursors of the same size. The large size of the precursor suggested that size-selection at mRNA or cDNA level may be necessary to obtain clones.

Example 6

cDNA Synthesis From the mRNA Preparations

cDNA synthesis was carried out using a kit from Amersham International. The first strand of the cDNA is synthesised by the enzyme reverse transcriptase, using the four nucleotide bases found in DNA (dATP, dTTP, dGTP, dCTP) and an oligo-dT primer. The second strand synthesis was by the method of Gubler and Hoffman (1983), whereby the RNA strand is nicked in many positions by RNase H, and the remaining fragments used to prime the replacement synthesis of a new DNA strand directed by the enzyme E. coli DNA polymerase I. Any 3' overhanging ends of DNA are filled in using the enzyme T4 polymerase. The whole process was monitored by adding a small proportion of ³² P!-dCTP into the initial nucleotide mixture, and measuring the percentage incorporation of label into DNA. Assuming that cold nucleotides are incorporated at the same rate, and that the four bases are incorporated equally, an estimate of the synthesis of cDNA can be obtained. From 1μg of mRNA approximately 140 ng of cDNA was synthesised. The products were analysed on an alkaline 1.4% agarose gel as described in the Amersham methods. Globin cDNA, synthesised as a control with the kit, was run on the same gel, which was dried down and autoradiographed. The cocoa cDNA had a range of molecular weights, with a substantial amount larger than the 600 bp of the globin cDNA.

Example 7

Cloning of cDNA into a Plasmid Vector by Homopolymer Tailing

The method of cloning cDNA into a plasmid vector was to 3' tail the cDNA with dC residues using the enzyme terminal transferase (Boehringer Corporation Ltd), and anneal into a PstI-cut and 5' tailed plasmid (Maniatis et al, 1982 Eschenfeldt et al. 1987). The optimum length for the dC tail is 12-20 residues. The tailing reaction (conditions as described by the manufacturers) was tested with a 1.5 kb blunt-ended restriction fragment, taking samples at intervals, and monitoring the incorporation of a small amount of ³² P!-dCTP. A sample of cDNA (70 ng) was then tailed using the predetermined conditions.

A dG-tailed plasmid vector (3'-oligo(dG)-tailed pUC9) was purchased from Pharmacia. 15 ng vector was annealed with 0.5-5 ng of cDNA at 58° C. for 2 hours in annealing buffer:5 mM Tris-HCl pH 7.6; 1 mM EDTA, 75 mM NaCl in a total volume of 50 μl. The annealed mixture was transformed into E. coli RRI (Bethesda Research Laboratories), transformants being selected on L-agar +100 μg/ml ampicillin. Approximately 200 transformants per ng of cDNA were obtained. Transformants were stored by growing in 100 1μl L-broth in the wells of microtitre plates, adding 100 μl 80% glycerol, and storing at -20° C.

Some of the dC tailed cDNA was size selected by electrophoresing on a 0.8% agarose gel, cutting slits in the gel at positions corresponding to 0.5, 1.0 and 1.5 kb, inserting DE81 paper and continuing electrophoresis until the cDNA had run onto the DE81 paper. The DNA was then eluted from the paper with high salt buffer, according to the method of Dretzen et al (1981).

Example 8

Construction of Oligonucleotide Probes for the 47131 kD Gene

The amino-acid sequence (SEQ ID NO:18) obtained from a cyanogen bromide peptide common to the 47 kD and 31 kD polypeptides is as follows:

Met-Phe-Glu-Ala-Asn-Pro-Asn-Thr-Phe

and the least redundant probe of 17 residues (a mixture of 32) (SEQ ID NO:19) is shown below: ##STR1## The actual probe was made anti-sense so that it could also be used to probe mRNA. Probe synthesis was carried out using an Applied Biosystems apparatus.

Example 9

Use of Oligonucleotides to Probe cDNA Library

The oligonucleotide probes were 5' end-labelled with gamma- ³² P! dATP and the enzyme polynucleotide kinase (Amersham International). The method was essentially that of Woods (1982, 1984), except that a smaller amount of isotope (15 μCi) was used to label about 40 ng probe in 10 mM MgCl₂, 100 mM Tris-HCl. pH 7.6; 20 mM 2-mercaptoethanol.

The cDNA library was grown on GeneScreen (New England Nuclear) nylon membranes placed on the surface of L-agar+100 μg/ml ampicillin plates. (The word GeneScreen is a trade mark.) Colonies were transferred from microtitre plates to the membranes using a 6×8 multi-pronged device, designed to fit into the wells of half the microtitre plate. Colonies were grown overnight at 37° C., lysed in sodium hydroxide and bound to membranes as described by Woods (1982, 1984). After drying the membranes were washed extensively in 3×SSC/0.1% SDS at 65° C. and hybridised to the labelled probe, using a HYBAID apparatus from Hybaid Ltd, PO Box 82, Twickenham, Middlesex. (The word HYBAID is a trade mark.) Conditions for hybridisation were as described by Mason & Williams (1985), a T_(d) being calculated for each oligonucleotide according to the formula:

    T.sub.d =4° C. per GC base pair+2° C. per AT base pair.

At mixed positions the lowest value is taken.

Hybridisation was carried out at T_(d) -5° C. Washing was in 6×SSC, 0.1% SDS initially at room temperature in the HYBAID apparatus, then at the hybridisation temperature (T_(d) -5° C.) for some hours, and finally at T_(d) for exactly 2 minutes. Membranes were autoradiographed onto FUJI X-ray film, with intensifying screens at-70° C. (The word FUJI is a trade mark.) After 24 -48 hours positive colonies stood out as intense spots against a low background.

Example 10

Analysis of Positive Clones for the 47 kD/31 kD Polypeptide

Only one positive clone. pMS600, was obtained. This released two PstI fragments on digestion of total length 1.3 kb, insufficient to encode the 67 kD precursor. The total insert was removed from the vector on a HindIII-EcoRI fragment, nick-translated and used to probe the cDNA library, picking up a further two positive clones. pMS700 and pMS800. Restriction mapping of all three inserts suggested an overlapping map covering nearly 2.0 kb, sufficient to encode the 67 kD precursor (see FIG. 1).

Example 11

Sequencing the Cloned Inserts

The sequencing strategy was to clone the inserts, and where appropriate subclones thereof, into the multiple cloning site of the plasmids pTZ18R/pTZ19R (Pharmacia). These plasmids are based on the better-known vectors pUC18/19 (Norrander et al. 1983), but contain a single-stranded origin of replication from the filamentous phage fl. When superinfected with phages in the same group, the plasmid is induced to undergo single-stranded replication, and the single-strands are packaged as phages extruded into the medium. DNA can be prepared from these `phages` using established methods for M13 phages (Miller, 1987), and used for sequencing by the method of Sanger (1977) using the reverse sequencing primer. The superinfecting phage used is a derivative of M13 termed M13K07, which replicates poorly and so does not compete well with the plasmid, and contains a selectable kanamycin-resistance marker. Detailed methods for preparing single-strands from the pTZ plasmids and helper phages are supplied by Pharmacia. DNA sequence was compiled and analysed using the Staden package of programs (Staden, 1986), on a PRIME 9955 computer. (The word PRIME is a trade mark.)

Example 12

Features of the 47 kD/31 kD cDNA and Deduced Amino-acid Sequence of the 67 kD Precursor

DNA sequencing of the three positive clones. pMS600, pMS700, pMS800, confirmed the overlap presumed in FIG. 1. No sequence differences were found in the overlapping regions (about 300 bp altogether), suggesting that the three cDNAs were derived from the same gene. The sequence of the combined cDNAs comprising 1818 bases is shown in FIG. 2. The first ATG codon is found at position 14, and is followed by an open reading frame of 566 codons. There is a 104-base 3' untranslated region containing a polyadenylation signal at position 1764. The oligonucleotide probe sequence is found at position 569.

The open reading frame translates to give a polypeptide of 566 amino-acids (FIG. 2), and a molecular weight of 65612, which is reasonably close to the 67 kD measured on SDS-PAGE gels. The N-terminal residues are clearly hydrophobic and look like a characteristic signal sequence. Applying the rules of Von Heije (1983), which predict cleavage sites for signal sequences, suggests a cleavage point between amino-acids 20 and 21 (see FIG. 3). The region following this is highly hydrophobic and contains four Cys-X-X-X-Cys motifs.

The N-terminus of the mature protein has been roughly identified as the glutamate (E) residue at 135 (FIG. 3), on the basis of some tentative N-terminal sequence (SEQ ID NO:3) (EEPGSQFANPAYHF). This N-terminus would give a mature protein of 49068 kD, in rough agreement with that observed. There appears to be no glycosylation sites (Asn-X-Ser/Thr) in the mature protein of the sequence.

Homologies Between the 67 kD Precursor and Other Known Proteins

Searches through the PIR database, and through the literature, revealed close homologies between the 67 kD polypeptide and a class of seed storage proteins termed vicilins, one of two major classes of globulins found in seed (Borroto and Dure, 1987). Alignments between the 67 kD polypeptide and vicilins from cotton (Gossypium hirsutum, Ghi), soybean (Glycine max, Gma), pea (Pisum sativum, Psa-c is convicilin. Psa-v is vicilin) and bean (Phaseolus vulgaris, Pvu) are shown in FIG. 4 (Bown et al, 1988; Chlan et al, 1986; Doyle et al, 1986; Lycett et al. 1983). Identical residues are boxed.

All the vicilins have a mature molecular weight of around 47 kD, with the exception of soybean conglycinin alpha and alphal subunits, which are 72 kD and 76 kD respectively, and pea convicilin with a mature molecular weight of 64kD. The pea and bean subunits (2 sub classes each) are synthesised as small precursors, around 50 kD). The most striking homology with the 67 kD is the cotton vicilin (Chlan et al, 1986). Cotton is also the most closely related to cocoa: both are members of the order Malvales. Interestingly cotton also has a large precursor, of 69 kD, comprising a short signal sequence, a large hydrophilic domain containing six Cys-X-X-X-Cys motifs, and a mature domain. It may therefore be possible to synthesise the corresponding cotton protein, by techniques analogous to those disclosed in this application and to use the cotton protein, or fragments of it, in the preparation of flavour components analogous to cocoa flavour components.

Example 13

Expression of the 67 kD Polypeptide in E. coli

Before the 67 kD coding region could be inserted into a expression vector the overlapping fragments from the three separate positive clones had to be spliced into a continuous DNA segment. The method of splicing is illustrated in FIG. 6: a HindIII-BglII fragment from pMS600, a BglII-EcoRI fragment from pMS700 and an EcoRI-SalI fragment from pMS800 were ligated into pTZ19R cut with HindIII and SalI. The resulting plasmid, containing the entire 67 kD cDNA, was termed pMS900.

An NcoI site was introduced at the ATG start codon, using the mutagenic primer: (SEQ ID NO:20)

5' TAG CAA CCA TGG TGA TCA 3'.

In vitro mutagenesis was carried out using a kit marketed by Amersham International, which used the method of Eckstein and co-workers (Taylor et al, 1985). After annealing the mutagenic primer to single-stranded DNA the second strand synthesis incorporates alpha-thio-dCTP in place of dCTP. After extension and ligation to form closed circles, the plasmid is digested with NciI, an enzyme which cannot nick DNA containing thio-dC. Thus only the original strand is nicked, and subsequently digested with exonuclease III. The original strand is then resynthesised, primed by the remaining DNA fragments and complementing the mutated position in the original strand. Plasmids are then transformed into E. coli and checked by plasmid mini preparations.

The 67 kD cDNA was then cloned into the E. coli expression plasmid, pJLA502 (FIG. 5), on an NcoI-SalI fragment (pMS902). pJLA502 (Schauder et al, 1987) is marketed by Medac GmbH, Postfach 303629, D-7000, Hamburg 36 and contains the strong lambda promoters, P_(L) and P_(R), and the leader sequence and ribosome binding site of the very efficiently translated E. coli gene, atpE. It also contains a temperature-sensitive cI repressor, and so expression is repressed at 30° C. and activated at 42° C. The vector has an NcoI site (containing an ATG codon: CCATGG) correctly placed with respect to the ribosome binding site, and foreign coding sequences must be spliced in at this point.

The expression vector was transformed into E. coli UT580. The transformed strain was grown in L-broth+ampicillin (100 μg/ml) at 30° C. until log phase (OD₆₁₀ =0.5) and the temperature was then shifted to 42° C. and samples taken at intervals. Samples were dissociated by boiling in SDS loading buffer, and run on SDS-PAGE gels. The proteins were electroblotted onto nitrocellulose membranes (Towbin et al. 1979) and Western blotting carried out using the anti-21 kD antibody prepared in Example 3 above (at 2 μg/ml) and as a secondary antibody, goat anti-rabbit-IgG conjugated to alkaline phosphatase (Scott et al, 1988).

A specific band at 67 kD was produced by pMS902, showing that a functional gene was present.

Example 14

Expression of the 67 kD Polypeptide in Yeast

Two yeast expression vectors were used, both based on a yeast-E. coli shuttle vector containing yeast and E. coli origins of replication, and suitable selectable markers (ampicillin-resistance for E. coli and leucine auxotrophy for yeast). Both vectors contain the yeast pyruvate kinase (PK) promoter and leader sequence and have a HindIII cloning site downstream of the promoter. One vector, A (YVA), is designed for internal expression, and the other, B (YVB), for secreted expression, having a portion of the signal sequence of the yeast mating alpha-factor downstream of the promoter, with a HindIII site within it to create fusion proteins with incoming coding sequences. The vectors are illustrated in FIG. 7.

To use the vectors effectively it is desirable to introduce the foreign coding region such that for vector A, the region from the HindIII cloning site to the ATG start is the same as the yeast PK gene, and for vector B, the remainder of the alpha-factor signal, including the lysine at the cleavage point. In practice this situation was achieved by synthesising two sets of HindIII- NcoI linkers to breach the gap between the HindIII cloning site in the vector and the NcoI at the ATG start of the coding sequence. This is illustrated in FIG. 8.

In order to use the yeast vector B, the hydrophobic signal sequence must first be deleted from the 67 kD cDNA. Although direct evidence of the location of the natural cleavage site was lacking, the algorithm of Von Heije predicts a site between amino-acids 20 (alanine) and 21 (leucine). However it was decided to remove amino-acids 2-19 by deletion, so that the useful NcoI site at the translation start would be maintained.

For ease of construction of the yeast vectors, the strategy was to first clone the HindIII- NcoI linkers into the appropriate pTZ plasmids, and then to clone the linkers plus coding region into the yeast vectors on HindIII-BamHI fragments. However the coding region contains an internal BamHI which must be removed by in vitro mutagenesis, giving a new plasmid pMS903. The signal sequence was deleted from pMS903 using the mutagenic primer (SEQ ID NO:21)

5'AGCATAGCAACCATGGTTGCTTTGTTCT 3'

to give pMS904. The appropriate HindIII- NcoI linkers were then cloned into pMS903 and pMS904 to give pMS907 and pMS905 respectively, and the HindIII- BamHI fragments (linkers+coding region) subcloned from these intermediate plasmids into YVA and YVB respectively to give the yeast expression plasmids pMS908 and pMS906. A diagrammatic scheme for these and other constructs is given in FIG. 9.

Because the mature cocoa protein appears to lack the N-terminal hydrophilic domain, as described in Example 12, expression vectors have also been designed to express the mature protein directly. Yeast is unlikely to have the same processing enzymes as cocoa and optimum expression may be obtained for a protein as close as possible to that found naturally in cocoa. Hence the DNA encoding the hydrophilic domain (amino acids 20-134) was deleted from the intermediate plasmids pMS907 and pMS905 to give plasmids pMS911 and pMS909 respectively, and the HindIII- BamHI fragments for these were cloned into YVA and YVB to give the expression plasmids pMS912 and pMS910 (FIG. 9).

A further modification was introduced by constructing expression in which the plant terminator had been removed and replaced with the yeast ADH terminator (present in YVA and YVB). The plant signal was removed by cutting the intermediate plasmids pMS907 and pMS905 at the PvuII site immediately downstream of the coding region, at position 1716 in FIG. 2. HindIII linkers were added and the entire coding region cloned into the yeast expression vectors on HindIII- HindIII fragments giving expression plasmids pMS914 (YVA) and pMS916 (YVB) (FIG. 9). A summary of the constructs made is given in FIG. 10.

The yeast expression plasmids were transferred into yeast spheroplasts using the method of Johnston (1988). The transformation host was the LEU³¹ strain AH22, and transformants were selected on leucine-minus minimal medium. LEU³⁰ transformants were streaked to single colonies, which were grown in 50 ml YEPD medium (Johnston. 1988) at 28|C for testing the extent and distribution of foreign protein. Cells were harvested from cultures in preweighed tubes in a bench-top centrifuge, and washed in 10 ml lysis buffer (200 mM Tris. pH 8.1:10% glycerol). The cell medium was reserved and concentrated 10-25×in an AMICON mini concentrator. (The word AMICON is a trade mark.) The washed cells were weighed and resuspended in lysis buffer plus protease inhibitors (1 mM phenyl methyl sulphonyl fluoride (PMSF); 1 μg/ml aprotinin; 0.5 μg/ml leupeptin) at a concentration of 1 g/ml. 1 volume acid-washed glass-beads was added and the cells broken by vortexing for 8 minutes in total, in 1 minute bursts, with 1 minute intervals on ice. After checking under the microscope for cell breakage, the mixture was centrifuged at 7000 rpm for 3 minutes to pellet the glass beads. The supernatant was removed to a pre-chilled centrifuge tube, and centrifuged for 1 hour at 20,000 rpm. (Small samples can be centrifuged in a microcentrifuge in the cold.) The supernatant constitutes the soluble fraction. The pellet was resuspended in 1 ml lysis buffer plus 10% SDS and 1% mercaptoethanol and heated at 90° C. for 10 minutes. After centrifuging for 15 minutes in a microcentrifuge the supernatant constitutes the particulate fraction.

Samples of each fraction and the concentrated medium were examined by Western blotting. Considering first the plasmids designed for internal expression in YVA, pMS908 produced immunoreactive proteins at 67 kD and 16 kD within the cells only. There was no evidence of the 67 kD protein being secreted under the influence of its own signal sequence. The smaller protein is presumed to be a degradation product. A similar result, but with improved expression, was obtained with pMS914, in which the plant terminator is replaced by a yeast terminator. However in pMS912, in which the coding region for the hydrophilic domain has been deleted, no synthesis of immunoreactive protein occurred.

For industrial production of heterologous proteins in yeast a secreted mode is preferable because yeast cells are very difficult to disrupt, and downstream processing from total cell protein is not easy. The results from the vectors constructed for secreted expressed were rather complicated. From the simplest construct, pMS906, in which the yeast α-factor signal sequence replaces the plant protein's own signal, immunoreactive proteins of approximately 47 kD, 28 kD and 18-20 kD were obtained and secreted into the medium. At first sight this is surprising because the coding region introduced should synthesise a 67 kD protein. However the most likely explanation is that the yeast's KEX2 protease, that recognises and cleaves the α-factor signal at a Lys-Arg site is also cleaving the 67 KD protein at Lys-Arg dipeptides at positions 148 and 313 in the amino-acid sequence. The calculated protein fragment sizes resulting from cleavage at these positions are 47179 Daltons, 28344 Daltons and 18835 Daltons, very close to the observed sizes.

When the plant terminator is replaced with a yeast terminator in pMS916 no expression is obtained in either cells or medium; it is possible that a mutation has been inadvertently introduced. From the construct pMS910, in which the hydrophilic domain has been deleted the main antigenic products were 28 KD and 18-20 kD, again secreted into the medium. It is presumed that the de novo 47 kD product is immediately cleaved at the KEX2 site at position 313.

In summary, four of the six expression vectors constructed direct the synthesis of proteins cross-reacting with anti-47 kD antibodies. Two of the constructs secrete proteins into the medium.

Example 15

Construction of Vectors Designed to Express the 67 kD Protein in Hansenula polymorpha

The methylotropic yeast Hansenula polymorpha offers a number of advantages over Saccharanyces cerevisiae as a host for the expression of heterologous proteins (EP-A-0173378 and Sudbery et al, 1988). The yeast will grow on methanol as sole carbon source, and under these conditions the enzyme methanol oxidase (MOX) can represent up to 40% of the total cell protein. Thus the MOX promoter is a very powerful one that can be used in a vector to drive the synthesis of heterologous proteins, and it is effective even as a single copy. This gives the potential to use stable integrated vectors. Hansenula can also grow on rich carbon sources such as glucose, in which case the MOX promoter is completely repressed. This means that cells containing the heterologous gene can be grown to a high density on glucose, and induced to produce the foreign protein by allowing the glucose to run out and adding methanol.

A plasmid, pHGL1, containing the MOX promoter and terminator, and a cassette containing the yeast α-factor secretory signal sequence, were prepared. The 67 kD coding region was cloned into pHGL1 on a BamHI--BamHI fragment, replacing the BglII fragment which contains the 3' end of the MOX coding region. The whole promoter--gene--terminator region can then be transferred to YEp13 on a BamHI--BamHI fragment to give the expression plasmid pMS922. The details of the construction are illustrated in FIG. 11. An analogous expression plasmid, pMS925, has been constructed with the yeast α-factor spliced onto the 67 kD coding region, replacing the natural plant signal. The BamHI-HindIII cassette containing the α-factor was ligated to the HindIII-BamHI fragment used to introduce the 67 kD coding region into YVB. The α-factor plus coding region was then cloned with pHGL1 on a BamHI--BamHI fragment, and transferred into YEP13 as before. Details are shown in FIG. 12.

Both constructs have been transformed into Hansenula and grown under inducing conditions with 0.5 % or 1% methanol. Both constructs directed the production of immunoreactive protein within the cells, and pMS925 secreted the protein into the medium under the influence of the α-factor signal sequence.

E. coli Strains

RR1 F³¹ v_(B) ³¹ M_(B) ara-14 proA2 leuB6 lacY1 galK2 vpsL20 (str^(r)) xyl-5 mtl-1 supE44³¹

CAG629 lac_(am) tvp_(am) Pho_(am) htpR_(am) mal rpsL lon supC_(ts)

UT580 (lac-pro) supE thi hsdD5/F'tra D36 proA³⁰ B³⁰ lacI^(q) lacZ M15

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    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 28                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1867 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Theobroma cacao                                                  (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 14..1711                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                       AAGCATAGCAAATATGGTGATCAGTAAGTCTCCTTTCATAGTTTTGATC49                            MetValIleSerLysSerProPheIleValLeuIle                                           1510                                                                           TTCTCTCTTCTCCTTTCTTTTGCGTTGCTTTGTTCTGGTGTCAGCGCC97                             PheSerLeuLeuLeuSerPheAlaLeuLeuCysSerGlyValSerAla                               152025                                                                         TATGGCAGAAAACAATATGAGCGTGATCCTCGACAGCAATACGAGCAA145                            TyrGlyArgLysGlnTyrGluArgAspProArgGlnGlnTyrGluGln                               303540                                                                         TGCCAGAGGCGATGCGAGTCGGAAGCGACTGAAGAAAGGGAGCAAGAG193                            CysGlnArgArgCysGluSerGluAlaThrGluGluArgGluGlnGlu                               45505560                                                                       CAGTGTGAACAACGCTGTGAAAGGGAGTACAAGGAGCAGCAGAGACAG241                            GlnCysGluGlnArgCysGluArgGluTyrLysGluGlnGlnArgGln                               657075                                                                         CAAGAAGAAGAGCTTCAAAGGCAATACCAGCAATGTCAAGGGCGTTGT289                            GlnGluGluGluLeuGlnArgGlnTyrGlnGlnCysGlnGlyArgCys                               808590                                                                         CAAGAGCAACAACAGGGGCAGAGAGAGCAGCAGCAGTGCCAGAGAAAA337                            GlnGluGlnGlnGlnGlyGlnArgGluGlnGlnGlnCysGlnArgLys                               95100105                                                                       TGCTGGGAGCAATATAAGGAACAAGAGAGAGGCGAGCACGAGAATTAC385                            CysTrpGluGlnTyrLysGluGlnGluArgGlyGluHisGluAsnTyr                               110115120                                                                      CATAATCACAAAAAAAATAGGAGCGAAGAAGAAGAAGGGCAACAAAGA433                            HisAsnHisLysLysAsnArgSerGluGluGluGluGlyGlnGlnArg                               125130135140                                                                   AACAATCCTTACTATTTTCCTAAAAGAAGATCATTCCAAACTCGATTC481                            AsnAsnProTyrTyrPheProLysArgArgSerPheGlnThrArgPhe                               145150155                                                                      AGGGATGAAGAGGGCAACTTCAAGATCCTCCAGAGGTTTGCTGAGAAC529                            ArgAspGluGluGlyAsnPheLysIleLeuGlnArgPheAlaGluAsn                               160165170                                                                      TCTCCTCCACTCAAGGGCATCAACGATTACCGCTTGGCCATGTTCGAA577                            SerProProLeuLysGlyIleAsnAspTyrArgLeuAlaMetPheGlu                               175180185                                                                      GCAAATCCCAACACTTTTATTCTTCCGCACCACTGTGATGCTGAGGCA625                            AlaAsnProAsnThrPheIleLeuProHisHisCysAspAlaGluAla                               190195200                                                                      ATTTACTTCGTGACAAACGGAAAGGGGACAATTACGTTTGTGACTCAT673                            IleTyrPheValThrAsnGlyLysGlyThrIleThrPheValThrHis                               205210215220                                                                   GAAAACAAAGAGTCCTATAATGTACAGCGTGGAACAGTAGTCAGCGTT721                            GluAsnLysGluSerTyrAsnValGlnArgGlyThrValValSerVal                               225230235                                                                      CCTGCAGGAAGCACTGTTTACGTGGTTAGCCAAGACAACCAAGAGAAG769                            ProAlaGlySerThrValTyrValValSerGlnAspAsnGlnGluLys                               240245250                                                                      CTAACCATAGCTGTGCTCGCCCTGCCTGTTAATTCTCCTGGCAAATAT817                            LeuThrIleAlaValLeuAlaLeuProValAsnSerProGlyLysTyr                               255260265                                                                      GAGTTATTCTTCCCCGCTGGAAATAATAAACCTGAATCATATTACGGA865                            GluLeuPhePheProAlaGlyAsnAsnLysProGluSerTyrTyrGly                               270275280                                                                      GCCTTCAGCTATGAAGTTCTTGAGACCGTCTTCAATACACAAAGAGAG913                            AlaPheSerTyrGluValLeuGluThrValPheAsnThrGlnArgGlu                               285290295300                                                                   AAGCTGGAGGAGATCTTGGAGGAACAGAGAGGGCAGAAGAGGCAGCAG961                            LysLeuGluGluIleLeuGluGluGlnArgGlyGlnLysArgGlnGln                               305310315                                                                      GGGCAGCAGGGTATGTTCCGGAAAGCCAAACCAGAGCAGATAAGAGCA1009                           GlyGlnGlnGlyMetPheArgLysAlaLysProGluGlnIleArgAla                               320325330                                                                      ATAAGCCAACAAGCTACTTCTCCAAGGCACAGAGGCGGGGAGAGACTT1057                           IleSerGlnGlnAlaThrSerProArgHisArgGlyGlyGluArgLeu                               335340345                                                                      GCCATCAATCTATTGAGCCAATCGCCTGTCTACTCCAACCAAAACGGA1105                           AlaIleAsnLeuLeuSerGlnSerProValTyrSerAsnGlnAsnGly                               350355360                                                                      CGCTTCTTTGAGGCTTGTCCTGAGGACTTCAGTCAATTTCAGAACATG1153                           ArgPhePheGluAlaCysProGluAspPheSerGlnPheGlnAsnMet                               365370375380                                                                   GATGTCGCTGTTTCAGCCTTCAAACTGAATCAGGGAGCCATATTTGTG1201                           AspValAlaValSerAlaPheLysLeuAsnGlnGlyAlaIlePheVal                               385390395                                                                      CCACACTACAATTCTAAGGCTACATTCGTGGTGTTTGTCACGGACGGA1249                           ProHisTyrAsnSerLysAlaThrPheValValPheValThrAspGly                               400405410                                                                      TATGGGTACGCTCAAATGGCTTGCCCGCATCTCTCCAGACAGAGCCAG1297                           TyrGlyTyrAlaGlnMetAlaCysProHisLeuSerArgGlnSerGln                               415420425                                                                      GGATCCCAAAGTGGAAGGCAAGACAGAAGAGAACAAGAAGAAGAGTCA1345                           GlySerGlnSerGlyArgGlnAspArgArgGluGlnGluGluGluSer                               430435440                                                                      GAAGAGGAGACATTTGGAGAATTCCAGCAGGTCAAAGCCCCATTGTCA1393                           GluGluGluThrPheGlyGluPheGlnGlnValLysAlaProLeuSer                               445450455460                                                                   CCTGGTGACGTCTTTGTAGCCCCGGCAGGCCATGCAGTTACATTCTTT1441                           ProGlyAspValPheValAlaProAlaGlyHisAlaValThrPhePhe                               465470475                                                                      GCATCCAAAGACCAGCCCCTGAATGCAGTTGCGTTTGGACTCAACGCC1489                           AlaSerLysAspGlnProLeuAsnAlaValAlaPheGlyLeuAsnAla                               480485490                                                                      CAGAACAACCAGAGAATTTTCCTTGCAGGGAAAAAGAACTTGGTCAGA1537                           GlnAsnAsnGlnArgIlePheLeuAlaGlyLysLysAsnLeuValArg                               495500505                                                                      CAAATGGATAGCGAGGCAAAGGAGTTATCATTTGGGGTACCATCGAAA1585                           GlnMetAspSerGluAlaLysGluLeuSerPheGlyValProSerLys                               510515520                                                                      TTGGTAGATAATATATTCAACAACCCGGATGAGTCGTATTTCATGTCT1633                           LeuValAspAsnIlePheAsnAsnProAspGluSerTyrPheMetSer                               525530535540                                                                   TTCTCTCAACAGAGGCAGCGTCGAGATGAAAGGAGGGGCAATCCCTTG1681                           PheSerGlnGlnArgGlnArgArgAspGluArgArgGlyAsnProLeu                               545550555                                                                      GCCTCAATTCTGGACTTTGCCCGCTTGTTCTAAGCAGCTGCTTCCACTTT1731                         AlaSerIleLeuAspPheAlaArgLeuPhe                                                 560565                                                                         TGTATCAGACATGCAGAGGCATGTAATGCAATAAATAAGTTGGCCTATGTAAAGAGGAGA1791               GAGTTTGCTTTTGTCTTGTTCTAACCTTGTTTTGAACTAGTAAACTTTCAATGTAATGAG1851               AGTTGTTATCTTTCTA1867                                                           (2) INFORMATION FOR SEQ ID NO: 2:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 566 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                       MetValIleSerLysSerProPheIleValLeuIlePheSerLeuLeu                               151015                                                                         LeuSerPheAlaLeuLeuCysSerGlyValSerAlaTyrGlyArgLys                               202530                                                                         GlnTyrGluArgAspProArgGlnGlnTyrGluGlnCysGlnArgArg                               354045                                                                         CysGluSerGluAlaThrGluGluArgGluGlnGluGlnCysGluGln                               505560                                                                         ArgCysGluArgGluTyrLysGluGlnGlnArgGlnGlnGluGluGlu                               65707580                                                                       LeuGlnArgGlnTyrGlnGlnCysGlnGlyArgCysGlnGluGlnGln                               859095                                                                         GlnGlyGlnArgGluGlnGlnGlnCysGlnArgLysCysTrpGluGln                               100105110                                                                      TyrLysGluGlnGluArgGlyGluHisGluAsnTyrHisAsnHisLys                               115120125                                                                      LysAsnArgSerGluGluGluGluGlyGlnGlnArgAsnAsnProTyr                               130135140                                                                      TyrPheProLysArgArgSerPheGlnThrArgPheArgAspGluGlu                               145150155160                                                                   GlyAsnPheLysIleLeuGlnArgPheAlaGluAsnSerProProLeu                               165170175                                                                      LysGlyIleAsnAspTyrArgLeuAlaMetPheGluAlaAsnProAsn                               180185190                                                                      ThrPheIleLeuProHisHisCysAspAlaGluAlaIleTyrPheVal                               195200205                                                                      ThrAsnGlyLysGlyThrIleThrPheValThrHisGluAsnLysGlu                               210215220                                                                      SerTyrAsnValGlnArgGlyThrValValSerValProAlaGlySer                               225230235240                                                                   ThrValTyrValValSerGlnAspAsnGlnGluLysLeuThrIleAla                               245250255                                                                      ValLeuAlaLeuProValAsnSerProGlyLysTyrGluLeuPhePhe                               260265270                                                                      ProAlaGlyAsnAsnLysProGluSerTyrTyrGlyAlaPheSerTyr                               275280285                                                                      GluValLeuGluThrValPheAsnThrGlnArgGluLysLeuGluGlu                               290295300                                                                      IleLeuGluGluGlnArgGlyGlnLysArgGlnGlnGlyGlnGlnGly                               305310315320                                                                   MetPheArgLysAlaLysProGluGlnIleArgAlaIleSerGlnGln                               325330335                                                                      AlaThrSerProArgHisArgGlyGlyGluArgLeuAlaIleAsnLeu                               340345350                                                                      LeuSerGlnSerProValTyrSerAsnGlnAsnGlyArgPhePheGlu                               355360365                                                                      AlaCysProGluAspPheSerGlnPheGlnAsnMetAspValAlaVal                               370375380                                                                      SerAlaPheLysLeuAsnGlnGlyAlaIlePheValProHisTyrAsn                               385390395400                                                                   SerLysAlaThrPheValValPheValThrAspGlyTyrGlyTyrAla                               405410415                                                                      GlnMetAlaCysProHisLeuSerArgGlnSerGlnGlySerGlnSer                               420425430                                                                      GlyArgGlnAspArgArgGluGlnGluGluGluSerGluGluGluThr                               435440445                                                                      PheGlyGluPheGlnGlnValLysAlaProLeuSerProGlyAspVal                               450455460                                                                      PheValAlaProAlaGlyHisAlaValThrPhePheAlaSerLysAsp                               465470475480                                                                   GlnProLeuAsnAlaValAlaPheGlyLeuAsnAlaGlnAsnAsnGln                               485490495                                                                      ArgIlePheLeuAlaGlyLysLysAsnLeuValArgGlnMetAspSer                               500505510                                                                      GluAlaLysGluLeuSerPheGlyValProSerLysLeuValAspAsn                               515520525                                                                      IlePheAsnAsnProAspGluSerTyrPheMetSerPheSerGlnGln                               530535540                                                                      ArgGlnArgArgAspGluArgArgGlyAsnProLeuAlaSerIleLeu                               545550555560                                                                   AspPheAlaArgLeuPhe                                                             565                                                                            (2) INFORMATION FOR SEQ ID NO: 3:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 14 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Theobroma cacao                                                  (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                           (B) LOCATION: 1..14                                                            (D) OTHER INFORMATION: /note= "Tentative N-terminal                            sequence of 47kD mature protein"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                       GluGluProGlySerGlnPheAlaAsnProAlaTyrHisPhe                                     1510                                                                           (2) INFORMATION FOR SEQ ID NO: 4:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 49 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: YEAST PYRUVATE KINASE GENE                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 41                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                       TTTACAAGACACCAATCAAAACAAATAAAACATCATCACAATGTCTAGA49                            MetSerArg                                                                      (2) INFORMATION FOR SEQ ID NO: 5:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                       MetSerArg                                                                      (2) INFORMATION FOR SEQ ID NO: 6:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 49 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: YEAST PYRUVATE KINASE GENE                                       (ix) FEATURE:                                                                  (A) NAME/KEY: mutation                                                         (B) LOCATION: 6..11                                                            (D) OTHER INFORMATION: /note= "Hin cloning site                                introduced"                                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 41                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                       TTTACAAGCTTCCAATCAAAACAAATAAAACATCATCACAATGTCTAGA49                            MetSerArg                                                                      (2) INFORMATION FOR SEQ ID NO: 7:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                       MetSerArg                                                                      1                                                                              (2) INFORMATION FOR SEQ ID NO: 8:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 33 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- feature                                              (B) LOCATION: 1..33                                                            (D) OTHER INFORMATION: /product="Hin-Nco linker"                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                       AGCTTCCAATCAAAACAAATAAAACATCATCAC33                                            (2) INFORMATION FOR SEQ ID NO: 9:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 33 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- feature                                              (B) LOCATION: 1..33                                                            (D) OTHER INFORMATION: /product="Hin-Nco linker"                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                       CATGGTGATGATGTTTTATTTGTTTTGATTGGA33                                            (2) INFORMATION FOR SEQ ID NO: 10:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 49 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 41                                                               (D) OTHER INFORMATION: /product="67kD expression vector                        A"                                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                      TTTACAAGCTTCCAATCAAAACAAATAAAACATCATCACCATGGTGATC49                            MetValIle                                                                      (2) INFORMATION FOR SEQ ID NO: 11:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                      MetValIle                                                                      1                                                                              (2) INFORMATION FOR SEQ ID NO: 12:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 27 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: YEAST ALPHA-FACTOR SIGNAL SEQUENCE                               (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..27                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                      GAAGGGGTAAGCTTGGATAAAAGAGAG27                                                  GluGlyValSerLeuAspLysArgGlu                                                    15                                                                             (2) INFORMATION FOR SEQ ID NO: 13:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 9 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                      GluGlyValSerLeuAspLysArgGlu                                                    15                                                                             (2) INFORMATION FOR SEQ ID NO: 14:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 17 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- feature                                              (B) LOCATION: 1..17                                                            (D) OTHER INFORMATION: /product="Hin-Nco linker"                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:                                      AGCTTGGATAAAAGAGC17                                                            (2) INFORMATION FOR SEQ ID NO: 15:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 17 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- feature                                              (B) LOCATION: 1..17                                                            (D) OTHER INFORMATION: /product="Hin-Nco linker"                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:                                      CATGGCTCTTTTATCCA17                                                            (2) INFORMATION FOR SEQ ID NO: 16:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..36                                                            (D) OTHER INFORMATION: /product="In phase fusion of 67kD                       coding region"                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:                                      GAAGGGGTAAGCTTGGATAAAAGAGCCATGGCGTTG36                                         GluGlyValSerLeuAspLysArgAlaMetAlaLeu                                           1510                                                                           (2) INFORMATION FOR SEQ ID NO: 17:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 12 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:                                      GluGlyValSerLeuAspLysArgAlaMetAlaLeu                                           1510                                                                           (2) INFORMATION FOR SEQ ID NO: 18:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 9 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..9                                                             (D) OTHER INFORMATION: /product="Cyanogen bromide peptide"                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:                                      MetPheGluAlaAsnProAsnThrPhe                                                    15                                                                             (2) INFORMATION FOR SEQ ID NO: 19:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 17 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- feature                                              (B) LOCATION: 1..17                                                            (D) OTHER INFORMATION: /product="N at position 6 = T or C                      N at position 9 = A or G                                                       N at position 12 = T,C,A or G                                                  N at position 15 = T or C"                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:                                      ATGTTNGANGCNAANCC17                                                            (2) INFORMATION FOR SEQ ID NO: 20:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:                                      TAGCAACCATGGTGATCA18                                                           (2) INFORMATION FOR SEQ ID NO: 21:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 28 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:                                      AGCATAGCAACCATGGTTGCTTTGTTCT28                                                 (2) INFORMATION FOR SEQ ID NO: 22:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 566 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Theobroma cacao                                                  (ix) FEATURE:                                                                  (A) NAME/KEY: Protein                                                          (B) LOCATION: 1..566                                                           (D) OTHER INFORMATION: /note= "67 kD Precursor Protein"                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:                                      MetValIleSerLysSerProPheIleValLeuIlePheSerLeuLeu                               151015                                                                         LeuSerPheAlaLeuLeuCysSerGlyValSerAlaTyrGlyArgLys                               202530                                                                         GlnTyrGluArgAspProArgGlnGlnTyrGluGlnCysGlnArgArg                               354045                                                                         CysGluSerGluAlaThrGluGluArgGluGlnGluGlnCysGluGln                               505560                                                                         ArgCysGluArgGluTyrLysGluGlnGlnArgGlnGlnGluGluGlu                               65707580                                                                       LeuGlnArgGlnTyrGlnGlnCysGlnGlyArgCysGlnGluGlnGln                               859095                                                                         GlnGlyGlnArgGluGlnGlnGlnCysGlnArgLysCysTrpGluGln                               100105110                                                                      TyrLysGluGlnGluArgGlyGluHisGluAsnTyrHisAsnHisLys                               115120125                                                                      LysAsnArgSerGluGluGluGluGlyGlnGlnArgAsnAsnProTyr                               130135140                                                                      TyrPheProLysArgArgSerPheGlnThrArgPheArgAspGluGlu                               145150155160                                                                   GlyAsnPheLysIleLeuGlnArgPheAlaGluAsnSerProProLeu                               165170175                                                                      LysGlyIleAsnAspTyrArgLeuAlaMetPheGluAlaAsnProAsn                               180185190                                                                      ThrPheIleLeuProHisHisCysAspAlaGluAlaIleTyrPheVal                               195200205                                                                      ThrAsnGlyLysGlyThrIleThrPheValThrHisGluAsnLysGlu                               210215220                                                                      SerTyrAsnValGlnArgGlyThrValValSerValProAlaGlySer                               225230235240                                                                   ThrValTyrValValSerGlnAspAsnGlnGluLysLeuThrIleAla                               245250255                                                                      ValLeuAlaLeuProValAsnSerProGlyLysTyrGluLeuPhePhe                               260265270                                                                      ProAlaGlyAsnAsnLysProGluSerTyrTyrGlyAlaPheSerTyr                               275280285                                                                      GluValLeuGluThrValPheAsnThrGlnArgGluLysLeuGluGlu                               290295300                                                                      IleLeuGluGluGlnArgGlyGlnLysArgGlnGlnGlyGlnGlnGly                               305310315320                                                                   MetPheArgLysAlaLysProGluGlnIleArgAlaIleSerGlnGln                               325330335                                                                      AlaThrSerProArgHisArgGlyGlyGluArgLeuAlaIleAsnLeu                               340345350                                                                      LeuSerGlnSerProValTyrSerAsnGlnAsnGlyArgPhePheGlu                               355360365                                                                      AlaCysProGluAspPheSerGlnPheGlnAsnMetAspValAlaVal                               370375380                                                                      SerAlaPheLysLeuAsnGlnGlyAlaIlePheValProHisTyrAsn                               385390395400                                                                   SerLysAlaThrPheValValPheValThrAspGlyTyrGlyTyrAla                               405410415                                                                      GlnMetAlaCysProHisLeuSerArgGlnSerGlnGlySerGlnSer                               420425430                                                                      GlyArgGlnAspArgArgGluGlnGluGluGluSerGluGluGluThr                               435440445                                                                      PheGlyGluPheGlnGlnValLysAlaProLeuSerProGlyAspVal                               450455460                                                                      PheValAlaProAlaGlyHisAlaValThrPhePheAlaSerLysAsp                               465470475480                                                                   GlnProLeuAsnAlaValAlaPheGlyLeuAsnAlaGlnAsnAsnGln                               485490495                                                                      ArgIlePheLeuAlaGlyLysLysAsnLeuValArgGlnMetAspSer                               500505510                                                                      GluAlaLysGluLeuSerPheGlyValProSerLysLeuValAspAsn                               515520525                                                                      IlePheAsnAsnProAspGluSerTyrPheMetSerPheSerGlnGln                               530535540                                                                      ArgGlnArgArgAspGluArgArgGlyAsnProLeuAlaSerIleLeu                               545550555560                                                                   AspPheAlaArgLeuPhe                                                             565                                                                            (2) INFORMATION FOR SEQ ID NO: 23:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 587 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Gossypium hirsutum                                               (ix) FEATURE:                                                                  (A) NAME/KEY: Protein                                                          (B) LOCATION: 1..587                                                           (D) OTHER INFORMATION: /note= "Vicilin from G. hirsutum"                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:                                      MetValArgAsnLysSerAlaCysValValLeuLeuPheSerLeuPhe                               151015                                                                         LeuSerPheGlyLeuLeuCysSerAlaLysAspPheProGlyArgArg                               202530                                                                         GlyAspAspAspProProLysArgTyrGluAspCysArgArgArgCys                               354045                                                                         GluTrpAspThrArgGlyGlnLysGluGlnGlnGlnCysGluGluSer                               505560                                                                         CysLysSerGlnTyrGlyGluLysAspGlnGlnArgHisArgProGlu                               65707580                                                                       AspProGlnArgArgTyrGluGluCysGlnGlnGluCysArgGlnGln                               859095                                                                         GluGluArgGlnArgProGlnCysGlnGlnArgCysIleLysArgPhe                               100105110                                                                      GluGlnGlnGlnGlnGlnSerGlnArgGlnPheGlnGluCysGlnGln                               115120125                                                                      HisCysHisGlnGlnGluGlnArgProGluArgLysGlnGlnCysVal                               130135140                                                                      AlaGluCysArgGluArgTyrGlnGluAsnProTrpArgArgGluArg                               145150155160                                                                   GluGluGluAlaGluGluGluGluThrGluGluGlyGluGlnGluGln                               165170175                                                                      SerHisAsnProPheHisPheHisArgArgSerPheGlnSerArgPhe                               180185190                                                                      ArgGluGluHisGlyAsnPheArgValLeuGlnArgPheAlaSerArg                               195200205                                                                      HisProIleLeuArgGlyIleAsnGluPheArgLeuSerIleLeuGlu                               210215220                                                                      AlaAsnProAsnThrGluValLeuProHisHisCysAspAlaGluLys                               225230235240                                                                   IleTyrLeuValThrAsnGlyArgGlyThrLeuThrPheLeuThrHis                               245250255                                                                      GluAsnLysGluSerTyrAsnValValProGlyValValValArgVal                               260265270                                                                      ProAlaGlySerThrValTyrLeuAlaAsnGlnAspAsnLysGluLys                               275280285                                                                      LeuIleIleAlaValLeuHisArgProValAsnAsnProArgGlnPhe                               290295300                                                                      GluGluPhePheProAlaGlySerGlnArgProGlnSerTyrLeuArg                               305310315320                                                                   AlaPheSerArgGluIleLeuGluProAlaPheAsnThrArgSerGlu                               325330335                                                                      GlnLeuAspGluLeuPheGlyGlyArgGlnSerHisArgArgGlnGln                               340345350                                                                      GlyGlnGlyMetPheArgLysAlaSerGlnGluGlnIleArgAlaLeu                               355360365                                                                      SerGlnGluAlaThrSerProArgGluLysSerGlyGluArgPheAla                               370375380                                                                      PheAsnLeuLeuTyrArgThrProArgTyrSerAsnGlnAsnGlyArg                               385390395400                                                                   PheTyrGluAlaCysProArgGluPheArgGlnLeuSerAspIleAsn                               405410415                                                                      ValThrValSerAlaLeuGlnLeuAsnGlnGlySerIlePheValPro                               420425430                                                                      HisTyrAsnSerLysAlaThrPheValValLeuValAsnGluGlyAsn                               435440445                                                                      GlyTyrValGluMetValSerProHisLeuProArgGlnSerSerPhe                               450455460                                                                      GluGluGluGluGluGlnGlnGlnGluGlnGluGlnGluGluGluArg                               465470475480                                                                   ArgSerGlyGlnTyrArgLysIleArgSerGlnLeuSerArgGlyAsp                               485490495                                                                      IlePheValValProAlaAsnPheProValThrPheValAlaSerGln                               500505510                                                                      AsnGlnAsnLeuArgMetThrGlyPheGlyLeuTyrAsnGlnAsnIle                               515520525                                                                      AsnProAspHisAsnGlnArgIlePheValAlaGlyLysIleAsnHis                               530535540                                                                      ValArgGlnTrpAspSerGlnAlaLysGluLeuAlaPheGlyValSer                               545550555560                                                                   SerArgLeuValAspGluIlePheAsnAsnAsnProGlnGluSerTyr                               565570575                                                                      PheValSerArgGlnArgGlnArgAlaSerGlu                                              580585                                                                         (2) INFORMATION FOR SEQ ID NO: 24:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 605 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Glycine max                                                      (ix) FEATURE:                                                                  (A) NAME/KEY: Protein                                                          (B) LOCATION: 1..605                                                           (D) OTHER INFORMATION: /note= "Vicilin from G. max"                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:                                      MetMetArgAlaArgPheProLeuLeuLeuLeuGlyLeuValPheLeu                               151015                                                                         AlaSerValSerValSerPheGlyIleAlaTyrTrpGluLysGluAsn                               202530                                                                         ProLysHisAsnLysCysLeuGlnSerCysAsnSerGluArgAspSer                               354045                                                                         TyrArgAsnGlnAlaCysHisAlaArgCysAsnLeuLeuLysValGlu                               505560                                                                         LysGluGluCysGluGluGlyGluIleProArgProArgProArgPro                               65707580                                                                       GlnHisProGluArgGluProGlnGlnProGlyGluLysGluGluAsp                               859095                                                                         GluAspGluGlnProArgProIleProPheProArgProGlnProArg                               100105110                                                                      GlnGluGluGluHisGluGlnArgGluGluGlnGluTrpProArgLys                               115120125                                                                      GluGluLysArgGlyGluLysGlySerGluGluGluAspGluAspGlu                               130135140                                                                      AspGluGluGlnAspGluArgGlnPheProPheProArgProProHis                               145150155160                                                                   GlnLysGluGluArgAsnGluGluGluAspGluAspGluGluGlnGln                               165170175                                                                      ArgGluSerGluGluSerGluAspSerGluLeuArgArgHisLysAsn                               180185190                                                                      LysAsnProPheLeuPhePheSerAsnArgPheGluThrLeuPheLys                               195200205                                                                      AsnGlnTyrGlyArgIleArgValLeuGlnArgPheAsnGlnArgSer                               210215220                                                                      ProGlnLeuGlnAsnLeuArgAspTyrArgIleLeuGluPheAsnSer                               225230235240                                                                   LysProAsnThrLeuLeuLeuProAsnHisAlaAspAlaAspTyrLeu                               245250255                                                                      IleValIleLeuAsnGlyThrAlaIleLeuSerLeuValAsnAsnAsp                               260265270                                                                      AspArgAspSerTyrArgLeuGlnSerGlyAspAlaLeuArgValPro                               275280285                                                                      SerGlyThrThrTyrTyrValValAsnProAspAsnAsnGluAsnLeu                               290295300                                                                      ArgLeuIleThrLeuAlaIleProValAsnLysProGlyArgPheGlu                               305310315320                                                                   SerPhePheLeuSerSerThrGluAlaGlnGlnSerTyrLeuGlnGly                               325330335                                                                      PheSerArgAsnIleLeuGluAlaSerTyrAspThrLysPheGluGlu                               340345350                                                                      IleAsnLysValLeuPheSerArgGluGluGlyGlnGlnGlnGlyGlu                               355360365                                                                      GlnArgLeuGlnGluSerValIleValGluIleSerLysGluGlnIle                               370375380                                                                      ArgAlaLeuSerLysArgAlaLysSerSerSerArgLysThrIleSer                               385390395400                                                                   SerGluAspLysProPheAsnLeuArgSerArgAspProIleTyrSer                               405410415                                                                      AsnLysLeuGlyLysPhePheGluIleThrProGluLysAsnProGln                               420425430                                                                      LeuArgAspLeuAspIlePheLeuSerIleValAspMetAsnGluGly                               435440445                                                                      AlaLeuLeuLeuProHisPheAsnSerLysAlaIleValIleLeuVal                               450455460                                                                      IleAsnGluGlyAspAlaAsnIleGluLeuValGlyLeuLysGluGln                               465470475480                                                                   GlnGlnGluGlnGlnGlnGluGluGlnProLeuGluValArgLysTyr                               485490495                                                                      ArgAlaGluLeuSerGluGlnAspIlePheValIleProAlaGlyTyr                               500505510                                                                      ProValValValAsnAlaThrSerAsnLeuAsnPhePheAlaIleGly                               515520525                                                                      IleAsnAlaGluAsnAsnGlnArgAsnPheLeuAlaGlySerGlnAsp                               530535540                                                                      AsnValIleSerGlnIleProSerGlnValGlnGluLeuAlaPhePro                               545550555560                                                                   GlySerAlaGlnAlaValGluLysLeuLeuLysAsnGlnArgGluSer                               565570575                                                                      TyrPheValAspAlaGlnProLysLysLysGluGluGlyAsnLysGly                               580585590                                                                      ArgLysGlyProLeuSerSerIleLeuArgAlaPheTyr                                        595600605                                                                      (2) INFORMATION FOR SEQ ID NO: 25:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 571 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Pisum sativum                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Protein                                                          (B) LOCATION: 1..571                                                           (D) OTHER INFORMATION: /note= "Convicilin from P. sativum"                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:                                      MetAlaThrThrValLysSerArgPheProLeuLeuLeuPheLeuGly                               151015                                                                         IleIlePheLeuAlaSerValCysValThrTyrAlaAsnTyrAspGlu                               202530                                                                         GlySerGluThrArgValProGlyGlnArgGluArgGlyArgGlnGlu                               354045                                                                         GlyGluLysGluGluLysArgHisGlyGluTrpArgProSerTyrGlu                               505560                                                                         LysGluGluHisGluGluGluLysGlnLysTyrArgTyrGlnArgGlu                               65707580                                                                       LysLysGluGlnLysGluValGlnProGlyArgGluArgTrpGluArg                               859095                                                                         GluGluAspGluGluGlnValGluGluGluTrpArgGlySerGlnArg                               100105110                                                                      ArgGluAspProGluGluArgAlaArgLeuArgHisArgGluGluArg                               115120125                                                                      ThrLysArgAspArgArgHisGlnArgGluGlyGluGluGluGluArg                               130135140                                                                      SerSerGluSerGlnGluHisArgAsnProPheLeuPheLysSerAsn                               145150155160                                                                   LysPheLeuThrLeuPheGluAsnGluAsnGlyHisIleArgArgLeu                               165170175                                                                      GlnArgPheAspLysArgSerAspLeuPheGluAsnLeuGlnAsnTyr                               180185190                                                                      ArgLeuValGluTyrArgAlaLysProHisThrIlePheLeuProGln                               195200205                                                                      HisIleAspAlaAspLeuIleLeuValValLeuAsnGlyLysAlaIle                               210215220                                                                      LeuThrValLeuSerProAsnAspArgAsnSerTyrAsnLeuGluArg                               225230235240                                                                   GlyAspThrIleLysIleProAlaGlyThrThrSerTyrLeuValAsn                               245250255                                                                      GlnAspAspGluGluAspLeuArgValValAspPheValIleProVal                               260265270                                                                      AsnArgProGlyLysPheGluAlaPheGlyLeuSerGluAsnLysAsn                               275280285                                                                      GlnTyrLeuArgGlyPheSerLysAsnIleLeuGluAlaSerLeuAsn                               290295300                                                                      ThrLysTyrGluThrIleGluLysValLeuLeuGluGluGlnGluLys                               305310315320                                                                   LysProGlnGlnLeuArgAspArgLysArgThrGlnGlnGlyGluGlu                               325330335                                                                      ArgAspAlaIleIleLysValSerArgGluGlnIleGluGluLeuArg                               340345350                                                                      LysLeuAlaLysSerSerSerLysLysSerLeuProSerGluPheGlu                               355360365                                                                      ProPheAsnLeuArgSerHisLysProGluTyrSerAsnLysPheGly                               370375380                                                                      LysLeuPheGluIleThrProGluLysLysTyrProGlnLeuGlnAsp                               385390395400                                                                   LeuAspIleLeuValSerCysValGluIleAsnLysGlyAlaLeuMet                               405410415                                                                      LeuProHisTyrAsnSerArgAlaIleValValLeuLeuValAsnGlu                               420425430                                                                      GlyLysGlyAsnLeuGluLeuLeuGlyLeuLysAsnGluGlnGlnGlu                               435440445                                                                      ArgGluAspArgLysGluArgAsnAsnGluValGlnArgTyrGluAla                               450455460                                                                      ArgLeuSerProGlyAspValValIleIleProAlaGlyHisProVal                               465470475480                                                                   AlaIleSerAlaSerSerAsnLeuAsnLeuLeuGlyPheGlyIleAsn                               485490495                                                                      AlaLysAsnAsnGlnArgAsnPheLeuSerGlySerAspAspAsnVal                               500505510                                                                      IleSerGlnIleGluAsnProValLysGluLeuThrPheProGlySer                               515520525                                                                      SerGlnGluValAsnArgLeuIleLysAsnGlnLysGlnSerHisPhe                               530535540                                                                      AlaSerAlaGluProGluGlnLysGluGluGluSerGlnArgLysArg                               545550555560                                                                   SerProLeuSerSerValLeuAspSerPheTyr                                              565570                                                                         (2) INFORMATION FOR SEQ ID NO: 26:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 410 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Pisum sativum                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Protein                                                          (B) LOCATION: 1..410                                                           (D) OTHER INFORMATION: /note= "Vicilin from P. sativum"                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:                                      MetLeuLeuAlaIleAlaPheLeuAlaSerValCysValSerSerArg                               151015                                                                         SerAspGlnGluAsnProPheIlePheLysSerAsnArgPheGlnThr                               202530                                                                         LeuTyrGluAsnGluAsnGlyHisIleArgLeuLeuGlnLysPheAsp                               354045                                                                         LysArgSerLysIlePheGluAsnLeuGlnAsnTyrArgLeuLeuGlu                               505560                                                                         TyrLysSerLysProHisThrLeuPheLeuProGlnTyrThrAspAla                               65707580                                                                       AspPheIleLeuValValLeuSerGlyLysAlaThrLeuThrValLeu                               859095                                                                         LysSerAsnAspArgAsnSerPheAsnLeuGluArgGlyAspAlaIle                               100105110                                                                      LysLeuProAlaGlySerIleAlaTyrPheAlaAsnArgAspAspAsn                               115120125                                                                      GluGluProArgValLeuAspLeuAlaIleProValAsnLysProGly                               130135140                                                                      GlnLeuGlnSerPheLeuLeuSerGlyThrGlnAsnGlnLysSerSer                               145150155160                                                                   LeuSerGlyPheSerLysAsnIleLeuGluAlaAlaPheAsnThrAsn                               165170175                                                                      TyrGluGluIleGluLysValLeuLeuGluGlnGlnGluGlnGluPro                               180185190                                                                      GlnHisArgArgSerLeuLysAspArgArgGlnGluIleAsnGluGlu                               195200205                                                                      AsnValIleValLysValSerArgAspGlnIleGluGluLeuSerLys                               210215220                                                                      AsnAlaLysSerSerSerLysLysSerValSerSerGluSerGlyPro                               225230235240                                                                   PheAsnLeuArgSerArgAsnProIleTyrSerAsnLysPheGlyLys                               245250255                                                                      PhePheGluIleThrProGluLysAsnGlnGlnLeuGlnAspLeuAsp                               260265270                                                                      IlePheValAsnSerValAspIleLysValGlySerLeuLeuLeuPro                               275280285                                                                      AsnTyrAsnSerArgAlaIleValIleValThrValThrGluGlyLys                               290295300                                                                      GlyAspPheGluLeuValGlyGlnArgAsnGluAsnGlnGlyLysGlu                               305310315320                                                                   AsnAspLysGluGluGluGlnGluGluGluThrSerLysGlnValGln                               325330335                                                                      LeuTyrArgAlaLysLeuSerProGlyAspValPheValIleProAla                               340345350                                                                      GlyHisProValAlaIleAsnAlaSerSerAspLeuAsnLeuIleGly                               355360365                                                                      LeuGlyIleAsnAlaGluAsnAsnGluArgAsnPheLeuAlaGlyGlu                               370375380                                                                      GluAspAsnValIleSerGlnValGluArgProValLysGluLeuAla                               385390395400                                                                   PheProGlySerSerHisGluValAspArg                                                 405410                                                                         (2) INFORMATION FOR SEQ ID NO: 27:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 421 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Phaseolus vulgaris                                               (ix) FEATURE:                                                                  (A) NAME/KEY: Protein                                                          (B) LOCATION: 1..421                                                           (D) OTHER INFORMATION: /note= "Vicilin from P. vulgaris"                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:                                      MetMetArgAlaArgValProLeuLeuLeuLeuGlyIleLeuPheLeu                               151015                                                                         AlaSerLeuSerAlaSerPheAlaThrSerLeuArgGluGluGluGlu                               202530                                                                         SerGlnAspAsnProPheTyrPheAsnSerAspAsnSerTrpAsnThr                               354045                                                                         LeuPheLysAsnGlnTyrGlyHisIleArgValLeuGlnArgPheAsp                               505560                                                                         GlnGlnSerLysArgLeuGlnAsnLeuGluAspTyrArgLeuValGlu                               65707580                                                                       PheArgSerLysProGluThrLeuLeuLeuProGlnGlnAlaAspAla                               859095                                                                         GluLeuLeuLeuValValArgSerGlySerAlaIleLeuValLeuVal                               100105110                                                                      LysProAspAspArgArgGluTyrPhePheLeuThrSerAspAsnPro                               115120125                                                                      IlePheSerAspHisGlnLysIleProAlaGlyThrIlePheTyrLeu                               130135140                                                                      ValAsnProAspProLysGluAspLeuArgIleIleGlnLeuAlaMet                               145150155160                                                                   ProValAsnAsnProGlnIleHisGluPhePheLeuSerSerThrGlu                               165170175                                                                      AlaGlnGlnSerTyrLeuGlnGluPheSerLysHisIleLeuGluAla                               180185190                                                                      SerPheAsnSerLysPheGluGluIleAsnArgValLeuPheGluGlu                               195200205                                                                      GluGlyGlnGlnGluGlyValIleValAsnIleAspSerGluGlnIle                               210215220                                                                      LysGluLeuSerLysHisAlaLysSerSerSerArgLysSerLeuSer                               225230235240                                                                   LysGlnAspAsnThrIleGlyAsnGluPheGlyAsnLeuThrGluArg                               245250255                                                                      ThrAspAsnSerLeuAsnValLeuIleSerSerIleGluMetGluGlu                               260265270                                                                      GlyAlaLeuPheValProHisTyrTyrSerLysAlaIleValIleLeu                               275280285                                                                      ValValAsnGluGlyGluAlaHisValGluLeuValGlyProLysGly                               290295300                                                                      AsnLysGluThrLeuGluTyrGluSerTyrArgAlaGluLeuSerLys                               305310315320                                                                   AspAspValPheValIleProAlaAlaTyrProValAlaIleLysAla                               325330335                                                                      ThrSerAsnValAsnPheThrGlyPheGlyIleAsnAlaAsnAsnAsn                               340345350                                                                      AsnArgAsnLeuLeuAlaGlyLysThrAspAsnValIleSerSerIle                               355360365                                                                      GlyArgAlaLeuAspGlyLysAspValLeuGlyLeuThrPheSerGly                               370375380                                                                      SerGlyAspGluValMetLysLeuIleAsnLysGlnSerGlySerTyr                               385390395400                                                                   PheValAspAlaHisHisHisGlnGlnGluGlnGlnLysGlyArgLys                               405410415                                                                      GlyAlaPheValTyr                                                                420                                                                            (2) INFORMATION FOR SEQ ID NO: 28:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 14 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:                                      GGAGACTGCCATGG14                                                               __________________________________________________________________________ 

We claim:
 1. An isolated and purified 67 kD protein of Theobroma cacao, having the amino acid sequence shown in FIG. 3 (SEQ ID NO:22).
 2. A protein as claimed in claim 1 which is recombinantly produced.
 3. An isolated and purified 47 kD protein of Theobroma cacao produced from a protein as claimed in claim
 2. 4. An isolated and purified 31 kD protein of Theobroma cacao produced from a protein as claimed in claim
 2. 5. Recombinant or isolated nucleic acid coding for a protein as claimed in claim
 1. 6. Nucleic acid as claimed in claim 5 which is DNA.
 7. Nucleic acid claimed in claim 5, which is in the form of a vector.
 8. Nucleic acid as claimed in claim 7, wherein the vector is an expression vector and the protein-coding sequence is operably linked to a promoter.
 9. Nucleic acid as claimed in claim 8, wherein the expression vector is a yeast expression vector and the promoter is a yeast pyruvate kinase (PK) promoter.
 10. Nucleic acid as claimed in claim 8, wherein the expression vector is a bacterial expression vector and the promoter is a strong lambda promoter.
 11. Nucleic acid as claimed in claim 8, comprising a signal sequence.
 12. A host cell comprising nucleic acid as claimed in claim
 7. 13. A host cell as claimed in claim 12 which is Saccharomyces cerevisiae.
 14. A host cell as claimed in claim 12 which is E. coli. 